The present invention relates generally to human chitinase enzyme and more specifically to novel purified and isolated polynucleotides encoding human chitinase, to the chitinase products encoded by the polynucleotides, to materials and methods for the recombinant production of chitinase products and to antibody substances specific for the chitinase.
Chitin is a linear homopolymer of xcex2-(1,4)-linked N-acetylglucosamine residues. This polysaccharide is second only to cellulose as the most abundant organic substance. The exoskeleton of arthropods is composed of chitin. In addition, fungi and other parasites contain chitin in their outer cell wall, where it serves important structural and protective roles. Disruption of the fungal cell wall and membrane has been a useful therapeutic strategy against fungi and parasites. For example, Amphotericin B and fluconazole exert their anti-fungal activity by affecting membrane steroids. Despite the existence of anti-fungal therapeutics, fungal infections of humans have increasingly become responsible for life-threatening disorders. See, Georgopapadakou et al., Trends Microbiol., 3: 98-104 (1995). The fungal species and parasites responsible for these diseases are mainly Canrda, Aspergillus, Cryptococcus, Histoplasma, Coccidioides and Pneumocystis. These pathogens are particularly dangerous in immunocompromised individuals, such as patients with AIDS, patients undergoing chemotherapy, and immunosuppressed organ transplant patients.
Chitin can be degraded by the enzyme chitinase. Chitinase enzymes are found in plants, microorganisms, and animals. Bacterial chitinase helps to provide a carbon source for bacterial growth. Insects produce chitinase to digest their cuticle at each molt. In plants, chitinase is thought to provide a protective role against parasitic fungi. Chitinases have been cloned from numerous bacterial [e.g., Serratia marcescens, Jones et al., EMBO J., 5:467-473 (1986)], plant [e.g., tobacco, Heitz et al., Mol. Gen. Genet., 245:246-254 (1994)], and insect [e.g., wasp, Krishnan et al., J. Biol. Chem., 269:20971-20976 (1994)] species.
Several proteins with low homology to bacterial, insect, and plant chitinases (less than 40% amino acid identity) have been identified in mammals, such as human cartilage gp-39 (C-gp39) [Hakala et al., J. Biol. Chem., 268.25803-25810 (1993)], human glycoprotein YKL-40 [Johansen et al., Eur. J. Cancer, 31A: 1437-1442 (1995)], oviduct-specific, estrogen-induced protein from sheep [DeSouza et al., Endocrinology, 136:2485-2496 (1995)], cows and humans; and a secretory protein from activated mouse macrophages [Chang et al., Genbank M94584]. However, chitin-degrading activity has not been reported for these proteins. The function of these proteins is not known, but they have been postulated to be involved in tissue remodeling. Hakala et al., supra, report that C-gp39 is detectable in synovial and cartilage specimens from rheumatoid arthritis patients, but not from normal humans. Recklies et al., Athritis Rheumatism, 36(9 SUPPL.):S190 (1993) report localization of the C-gp39 protein to a distinct population of cells in the superficial layers of cartilage. Johansen et al., supra, report that measurements of YKL-40 serum levels are of value as a potential prognostic marker for the extent of metastatic disease and survival of patients with recurrent breast cancer.
Escott et al., Infect. Immun., 63:4770-4773 (1995) demonstrated chitinase enzymatic activity in human leukocytes and in human serum. Overdijk et al., Glycobiology, 4:797-803 (1994) described isolation of a chitinase (4-methylumbelliferyl-tetra-N-acetylchitotetraoside hydrolase) from human serum and rat liver. Renkema et al., J. Biol. Chem., 270:2198-2202 (February 1995) prepared a human chitotriosidase from the spleen of a Gaucher disease patient. Their preparation exhibited chitinase activity and the article reports a small amount of amino acid sequence of the protein component of the preparation (22 amino terminal residues and 21 residues of a tryptic fragment). The function of human chitinase is also unknown, but a relationship with the pathophysiology of Gaucher disease is proposed in the article. A later publication by the same group [Boot et al., J. Biol. Chem., 270(44):26252-26256 (November 1995)] describes the cloning of a human macrophage cDNA encoding a product that exhibits chitinase activity. The partial amino acid sequence reported by the group in their February 1995 article matches portions of the deduced amino acid sequence of the human macrophage cDNA product. See also International Patent Publication No. WO 96/40940.
In view of the increasing incidence of life-threatening fungal infection in immunocompromised individuals, there exists a need in the art to identify and isolate polynucleotide sequences encoding human chitinases, to develop materials and methods useful for the recombinant production of the enzyme, and to generate reagents for the detection of the chitinase in plasma.
The present invention provides novel purified and isolated polynucleotides (i.e., DNA and RNA, both sense and antisense strands) encoding human chitinase or fragments and analogs thereof; methods for the recombinant production of chitinase polypeptides, fragments and analogs thereof; purified and isolated chitinase polypeptide fragments and analogs; antibodies to such polypeptides, fragments and analogs; and pharmaceutical compositions comprising these polypeptides, fragments, analogs, or antibodies.
Specifically provided are: purified, isolated polynucleotides encoding the human chitinase amino acid sequence of SEQ ID NOS: 2 or 4, particularly amino acids 1 to 445 thereof; DNAs comprising the protein coding nucleotides of SEQ ID NOS: 1 or 3, particularly nucleotides 65 to 1402 of SEQ ID NO: 1 or nucleotides 90 to 1427 of SEQ ID NO: 3; purified, isolated polynucleotides comprising a polynucleotide sequence encoding the amino acid sequence of SEQ ID NOS: 14 or 15; purified, isolated polynucleotides encoding human chitinase selected from the group consisting of: (a) a double-stranded DNA comprising the protein coding portions of the sequence set out in either SEQ ID NO: 1 or SEQ ID NO: 3, (b) a DNA which hybridizes under stringent conditions to a non-coding strand of the DNA of (a), and (c) a DNA which, but for the redundancy of the genetic code, would hybridize under stringent conditions to a non-coding strand of DNA sequence of (a) or (b); vectors comprising such DNAs, particularly expression vectors wherein the DNA is operatively linked to an expression control DNA sequence; host cells stably transformed or transfected with such DNAs in a manner allowing the expression in said host cell of human chitinase; a method for producing human chitinase comprising culturing such host cells in a nutrient medium and isolating human chitinase from said host cell or said nutrient medium; purified, isolated polypeptides produced by this method; purified, isolated polypeptides comprising the human chitinase amino acid sequence of SEQ ID NOS: 2 or 4, particularly amino acids 1 to 445 thereof; human chitinase fragments lacking from 1 to about 72 C-terminal amino acid residues of mature human chitinase, particularly the human chitinase fragment of SEQ ID NO: 14; the human chitinase analog of SEQ ID NO: 15; hybridoma cell lines producing a monoclonal antibody that is specifically reactive with one of the above-described polypeptides; and monoclonal antibodies produced by such hybridomas.
Preferred DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences. The nucleotide sequence of two human cDNAs encoding presumed allelic variants of human chitinase, and including noncoding 5xe2x80x2 and 3xe2x80x2 sequences, are set forth in SEQ ID NO: 1 and SEQ ID NO: 3. These DNA sequences and DNA sequences which hybridize to the noncoding strand thereof under standard stringent conditions or which would hybridize but for the redundancy of the genetic code, are contemplated by the invention. Preferred DNAs of the present invention comprise the human chitinase coding region (corresponding to nucleotides 2 to 1402 of SEQ ID NO: 1 or nucleotides 27 to 1427 of SEQ ID NO: 3), and the putative coding sequence of the mature, secreted human chitinase protein without its signal sequence (nucleotides 65 to 1402 of SEQ ID NO: 1, or nucleotides 90 to 1427 of SEQ ID NO: 3).
Exemplary stringent hybridization conditions are as follows: hybridization at 42xc2x0 C. in 50% formamide and washing at 60xc2x0 C. in 0.1xc3x97SSC, 0.1% SDS. It is understood by those of skill in the art that variation in these conditions occurs based on the length and GC nucleotide base content of the sequences to be hybridized. Formulas standard in the art are appropriate for determining exact hybridization conditions. See Sambrook et al., 9.47-9.51 in Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
Two amino acid sequences for human chitinase(s) are set forth in SEQ ID NOS: 2 and 4. The sequence of SEQ ID NO: 2 is encoded by the nucleotide sequence of SEQ ID NO: 1, and SEQ ID NO: 4 is encoded by the nucleotide sequence of SEQ ID NO: 3. Preferred polynucleotides of the present invention include, in addition to those polynucleotides described above, polynucleotides that encode amino acids xe2x88x9221 to 445 of SEQ ID NO: 2 or SEQ ID NO: 4, and that differ from the polynucleotides described in the preceding paragraphs only due to the well-known degeneracy of the genetic code. Similarly, since twenty-one amino acids (positions xe2x88x9221 to xe2x88x921) of SEQ ID NOS: 2 and 4 comprise a signal peptide that is cleaved to yield the mature human chitinase protein, preferred polynucleotides include those encoding polypeptides comprising amino acids 1 to 445 of SEQ ID NO: 2 or SEQ ID NO: 4.
Among the uses for the polynucleotides of the present invention is use as a hybridization probe, to identify and isolate genomic DNA encoding human chitinase; to identify and isolate non-human genes encoding proteins homologous to human chitinase; to identify human and non-human proteins having similarity to human chitinase (including those that may be involved in tissue remodeling); and to identify those cells which express human chitinase and the biological conditions under which this protein is expressed.
In another aspect, the invention includes biological replicas (i.e., copies of isolated DNA sequences made in vivo or in vitro) of DNA sequences of the invention. Autonomously replicating recombinant constructions such as plasmid and viral DNA vectors incorporating chitinase polynucleotides, including any of the DNAs described above, are provided. Preferred vectors include expression vectors in which the incorporated chitinase-encoding cDNA is operatively linked to an endogenous or heterologous expression control sequence and a transcription terminator. Such expression vectors may further include polypeptide-encoding DNA sequences operably linked to the chitinase-encoding DNA sequences, which vectors may be expressed to yield a fusion protein comprising the polypeptide of interest.
According to another aspect of the invention, procaryotic or eucaryotic host cells are stably transformed or transfected with DNA sequences of the invention in a manner allowing the desired chitinase product to be expressed therein. Host cells expressing chitinase products can serve a variety of useful purposes. Such cells constitute a valuable source of immunogen for the development of antibody substances specifically immunoreactive with chitinase. Host cells of the invention are useful in methods for the large scale production of chitinase wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated, e.g., by immunoaffinity purification, from the cells or from the medium in which the cells are grown.
Chitinase products may be obtained as isolates from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving procaryotic or eucaryotic host cells of the invention. Chitinase products of the invention may be full length polypeptides, fragments or analogs thereof. Chitinase products having part or all of the amino acid sequence set out in SEQ ID NO: 2 or SEQ ID NO: 4 are contemplated. One preferred fragment which lacks the C-terminal seventy-two amino acid residues of the mature protein is set forth in SEQ ID NO: 14. It has been determined that these seventy-two C-terminal residues are not critical to chitinase enzymatic activity. Example 5 illustrates production of this C-terminal fragment; the introduction of a stop codon after the codon for amino acid 373 resulted in a recombinant chitinase fragment of about 39 kDa that retained similar specific activity when compared with full length recombinant human chitinase.
Analogs may comprise chitinase analogs wherein one or more of the specified (i.e., naturally encoded) amino acids is deleted or replaced or wherein one or more nonspecified amino acids are added: (1) without loss of one or more of the enzymatic activities or immunological characteristics specific to chitinase; or (2) with specific disablement of a particular biological activity of chitinase. Example 3 illustrates the production of such an analog (SEQ ID NO: 15), in which the proline at position 370 is substituted with a serine, and in which the C-terminal seventy-two amino acid residues have been deleted. The use of mammalian host cells is also expected to provide for post-translational modifications (e.g., myristolation, glycosylation, truncation, lipidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention.
Proteins or other molecules that bind to chitinase may be used to modulate its activity. Also comprehended by the present invention are antibody substances (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, CDR-grafted antibodies and the like) and other binding proteins specific for chitinase. Proteins or other molecules (e.g., small molecules) which specifically bind to chitinase can be identified using chitinase isolated from plasma, recombinant chitinase, chitinase variants or cells expressing such products. Binding proteins are useful, in turn, in compositions for immunization as well as for purifying c.hitinase, and are useful for detection or quantification of chitinase in fluid and tissue samples by known immunological procedures. Anti-idiotypic antibodies specific for chitinase-specific antibody substances are also contemplated.
The scientific value of the information contributed through the disclosures of DNA and amino acid sequences of the present invention is manifest. As one series of examples, knowledge of the sequence of a cDNA for chitinase makes possible the isolation by DNA/DNA hybridization of genomic DNA sequences encoding chitinase and chitinase expression control regulatory sequences such as promoters, operators and the like. DNA/DNA hybridization procedures carried out with DNA sequences of the invention under conditions of stringency standard in the art are likewise expected to allow the isolation of DNAs encoding human allelic variants of chitinase, other structurally related human proteins sharing one or more of the biochemical and/or immunological properties of chitinase, and non-human species proteins homologous to chitinase. The DNA sequence information provided by the present invention also makes possible the development, by homologous recombination or xe2x80x9cknockoutxe2x80x9d strategies [see, e.g., Kapecchi, Science, 244: 1288-1292 (1989)], of rodents that fail to express a functional chitinase enzyme, overexpress chitinase enzyme, or express a variant chitinase enzyme. Polynucleotides of the invention when suitably labelled are useful in hybridization assays to detect the capacity of cells to synthesize chitinase. Polynucleotides of the invention may also be the basis for diagnostic methods useful for identifying a genetic alteration(s) in the chitinase locus that underlies a disease state or states. Also made available by the invention are anti-sense polynucleotides relevant to regulating expression of chitinase by those cells which ordinarily express the same.
Administration of chitinase preparations of the invention to mammalian subjects, especially humans, for the purpose of ameliorating disease states caused by chitin-containing parasites such as fungi is contemplated by the invention. Fungal infections (mycoses) such as candidiasis, aspergillosis, coccidioidomycosis, blastomycosis, paracoccidioidomycosis, histoplasmosis, cryptococcosis, chromoblastomycosis, sporotrichosis, mucormycosis, and the dermatophytoses can manifest as acute or chronic disease. Pathogenic fungi cause serious, often fatal disease in immunocompromised hosts. Cancer patients undergoing chemotherapy, immunosuppressed individuals, and HIV-infected individuals are susceptible to mycoses caused by Candida, Aspergillus, Pneumocystis carinii, and other fungi. Amphotericin B and fluconazole are useful therapeutics for fungal infections, but toxicity associated with these drugs causes serious adverse side effects that limit their usefulness. The mortality of systemic candidiasis is greater than 50% despite Amphotericin B treatment. Therefore, a need exists for agents that inhibit fungal growth in vivo; and such products may be used as single agents or in combination with currently approved, conventional anti-fungal compounds. Because growing fungi require chitin synthesis for survival, inhibition by recombinant human chitinase may be useful for limiting fungal infections in vivo. Animal models for fungal infection are illustrated below in Examples 8 through 14 and have been described in the art.
Specifically contemplated by the invention are chitinase compositions for use in methods for treating a mammal susceptible to or suffering from fungal infections comprising administering chitinase to the mammal in an amount sufficient to supplement endogenous chitinase activity. It is contemplated that the chitinase may be administered with other conventional anti-fungal agents, including amphotericin B and the structurally related compounds nystatin and pimaricin; 5-fluorocytosine; azole derivatives such as fluconazole, ketoconazole, clotrimazole, miconazole, econazole, butoconazole, oxiconazole, sulconazole, terconazole, itraconazole and tioconazole; allylamines-thiocarbamates, such as tolnaftate, naftifine and terbinafme; griseofulvin; ciclopirox olamine; haloprogin; undecylenic acid; and benzoic acid. [See, e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, 9th ed., McGraw-Hill, NY (1996).] Chitinase may improve the effectiveness of these conventional anti-fungal agents, perhaps by rendering the yeast more susceptible to their action, even in situations where the chitinase alone is not effective for inhibiting growth of fungi. By reducing the amount of conventional anti-fungal agent needed to exert the desired therapeutic effect, chitinase may allow the drugs to be used at less toxic levels. For example, Davies and Pope, Nature, 273:235-236 (1978) reported that administration of mycolases (enzymes that degrade the fungal cell wall) in conjunction with a normally ineffective dose of anti-fungal drug to Aspergillus-infected mice provided synergistically effective treatment. The combination of fungal chitinase and laminarinase was noted to be more effective in attacking the fungal cell wall than either enzyme alone.
Thus, the invention contemplates the use of chitinase in the preparation of a medicament for the prophylactic or therapeutic treatment of fungal infections, and further contemplates the use of chitinase in the preparation of a medicament for co-administration with another anti-fungal agent.
Therapeutic/pharmaceutical compositions contemplated by the invention include chitinase and a physiologically acceptable diluent or carrier and may also include other anti-fungal agents. Dosage amounts indicated would be sufficient to supplement endogenous chitinase activity. For general dosage considerations see Remington: The Science and Practice of Pharmacy, 19th ed., Mack Publishing Co., Easton, Pa. (1995). Dosages will vary between about 1 gg/kg to 100 mg/kg body weight, and preferably between about 0.1 to about 20 mg chitinase/kg body weight. Therapeutic compositions of the invention may be administered by various routes depending on the infection to be treated, including via subcutaneous, intramuscular, intravenous, intrapulmonary, transdermal, intrathecal, topical, oral, or suppository administration.
The invention also contemplates that the overexpression of chitinase in Gaucher disease or at sites of inflammation (such as in rheumatoid arthritis) may have deleterious effects on the extracellular matrix and, in such disease settings, inhibitors of chitinase activity may provide therapeutic benefit, e.g. by reducing remodeling or destruction of the extracellular matrix.
The human chitinase cDNA of the present invention was isolated from a macrophage cDNA library. Macrophages are known to be closely associated with rheumatoid arthritis lesions [Feldman et al., Cell, 85:307-310 (1996)], and macrophage products such as TNF-xcex1 are implicated in disease progression. A protein with homology to human chitinase, C-gp39, has been detected in the synovium and cartilage of rheumatoid arthritis patients. While the natural substrate for human chitinase is probably chitin from pathogenic organisms, the enzyme may also exhibit activity on endogenous macromolecules which form the natural extraceflular matrix. For example, it has been suggested that hyaluronic acid, a major component of the extracellular matrix, contains a core of chitin oligomers. [Semino et al., Proc. Nat""l Acad. Sci., 93:4548-4553 (1996); Varki, Proc. Nat""l. Acad. Sci., 93:4523-4525 (1996).] Chitinase may therefore be involved in degradation of extracellular matrix in diseases such as rheumatoid arthritis. The role of chitinase may be determined by measuring chitinase levels and/or the effects of chitinase administration or chitinase inhibition in synovial fluid isolated from arthritic joints. Endogenous chitinase levels can be measured by enzymatic assay or with an antibody. Viscosity of synovial fluid can be measured before and after chitinase treatment; a decrease of viscosity associated with chitinase would be consistent with an endogenous chitinase substrate. Modulation of chitinase activity could thereby modulate the progression of joint destruction in rheumatoid arthritis.
Also contemplated by the invention are methods for screening for inhibitors of chitinase activity, which may be useful in the manner described in the preceding paragraph. A method for screening samples to identify agents that inhibit chitinase is reported in, e.g., WO 95/34678 published Dec. 21, 1995.
Further contemplated are methods for measuring endogenous levels of chitinase, e.g., for diagnosing Gaucher""s disease. Hollak et al., J. Clin. Invest., 93:1288-1292 (1994), report that plasma chitinase levels are a diagnostic marker for Gaucher""s disease. The recombinant proteins of this invention are expected to be more useful than preparations purified from humans, which have associated problems of yield and contamination with other impurities or infectious agents.
Other aspects and advantages of the present invention will be understood upon consideration of the following illustrative examples. Example 1 describes the isolation of human chitinase cDNA clones from a human macrophage cDNA library. Example 2 addresses the pattern of chitinase gene expression in various human tissues. Example 3 describes the recombinant expression of the human chitinase gene in prokaryotic cells and purification of the resulting enzyme. Example 4 provides a protocol for the recombinant production of human chitinase in yeast. Example 5 describes the recombinant expression of the human chitinase gene in mammalian cells and purification of the resulting protein. Example 6 describes production of human chitinase polypeptide analogs by peptide synthesis or recombinant production methods. Example 7 provides a protocol for generating monoclonal antibodies that are specifically immunoreactive with human chitinase. Example 8 describes an assay for the measurement of chitinase catalytic activity. Example 9 addresses determination of the anti-fungal activity of human chitinase in vitro. Example 10 addresses determination of the anti-fungal activity of human chitinase in vivo in a mouse model, and Examples 11 through 14 address rabbit models of invasive aspergillosis, disseminated candidiasis, Candida ophthalmitis, and Candida endocarditis.